The present invention relates to new chemical compounds which stimulate soluble guanylate cyclase, their preparation and their use as medicaments, in particular as medicaments for the treatment of cardiovascular diseases.
One of the most important cellular transmission systems in mammals is cyclic guanosine monophosphate (cGMP). Together with nitrogen monoxide (NO) which is released from the endothelium and transmits hormonal and mechanical signals, it forms the NO/cGMP system. The guanylate cyclases catalyse the biosynthesis of cGMP from guanosine triphosphate (GTP). The representatives of this family known hitherto can be divided into two groups according to structural features and according to the nature of the ligands: the particulate guanylate cyclases, which can be stimulated by natriuretic peptides, and the soluble guanylate cyclases, which can be stimulated by NO. The soluble guanylate cyclases consist of two subunits and very highly probably contain one haem per heterodimer, which is a part of the regulatory centre. This has a central importance for the activation mechanism. NO can bind to the iron atom of the haem and thus markedly increase the activity of the enzyme. Haem-free preparations on the other hand, cannot be stimulated by NO. CO is also able to attack at the central iron atom of the haem, where the stimulation by CO is markedly lower than that by NO.
Owing to the formation of cGMP and the regulation of phosphodiesterases, iron channels and protein kinases resulting therefrom, guanylate cyclase plays a crucial role in different physiological processes, in particular in the relaxation and proliferation of smooth muscle cells, platelet aggregation and adhesion and neuronal signal transmission, and in diseases which are based on a disturbance of the abovementioned processes. Under pathophysiological conditions, the NO/cGMP system may be suppressed, which can lead, for example, to high blood pressure, platelet activation, increased cell proliferation, endothelial dysfunction, atherosclerosis, angina pectoris, cardiac insufficiency, thromboses, stroke and myocardial infarct.
An NO-independent possibility of treatment for diseases of this type, which is targeted at influencing the cGMP signal pathway in organisms, is a promising approach on account of the high efficiency and low side effects which are to be expected.
For therapeutic stimulation of the soluble guanylate cyclase, hitherto exclusively compounds such as organic nitrates have been used, whose action is based on NO. This is formed by bioconversion and activates the soluble guanylate cyclase by attacks on the central iron atom of the haem. Besides the side effects, the crucial disadvantages of this manner of treatment includes the development of tolerance.
In recent years, a few substances have been described which stimulate soluble guanylate cyclase directly, i.e. without prior release of NO, for example 3-(5xe2x80x2-hydroxymethyl-2xe2x80x2-furyl)-1-benzylindazole (YC-1, Wu et al., Blood 84 (1994), 4226; Mxc3xclsch et al., Br. J. Pharmacol. 120 (1997), 681), Fatty acids (Goldberg et al., J. Biol. Chem. 252 (1977), 1279), diphenyliodonium-hexafluorophosphate (Pettibone et al., Eur. J. Pharmacol. 116 (1985), 307), isoliquiritigenin (Yu et al., Brit. J. Pharmacol. 114 (1995), 1587) and various substituted pyrazole derivatives (WO 98/16223).
Furthermore, pyrazolopyridine derivatives have been described as stimulators of soluble guanylate cyclase in WO 98/16507, WO 98/23619, WO 00/06567, WO 00/06568, WO 00/06569 and WO 00/21954. In these patent applications, pyrazolopyridines are also described which have a pyrimidine radical in the 3 position. Compounds of this type have a very high in vitro activity with respect to the stimulation of soluble guanylate cyclase. However, it has been seen that these compounds have some disadvantages with respect to their in vivo properties, for example their behaviour in the liver, their pharmacological behaviour, their dose-response relationship or their metabolization pathway.
It was therefore the object of the present invention to provide further pyrazolopyridine derivatives which act as stimulators of soluble guanylate cyclase, but which do not have the abovementioned disadvantages of the compounds from the prior art.
This object is achieved according to the present invention by the compounds according to claim 1. These new pyrazolopyridine derivatives are distinguished by a pyrimidine radical in the 3 position which has a certain substitution pattern, namely a pyridine radical in the 5 position of the pyrimidine ring and an amino group in the 4 position of the pyrimidine ring.
Specifically, the present invention relates to the compounds of the formula (I) 
in which
R1 represents 4-pyridinyl or 3-pyridinyl;
R2 represents H, NH2 or halogen;
and salts, isomers and hydrates thereof.
According to an alternative embodiment the present invention relates to the compounds of the formula (I), wherein
R1 represents 4-pyridinyl or 3-pyridinyl;
R2 represents H, NH2 or Cl;
and salts, isomers and hydrates thereof.
According to a further alternative embodiment the present invention relates to the compounds of the formula (I), wherein
R1 represents 4-pyridinyl or 3-pyridinyl;
R2 represents H;
and salts, isomers and hydrates thereof.
The compounds of the formula (I) according to the invention can also be present in the form of their salts. In general, salts with organic or inorganic bases or acids may be mentioned here.
In the context of the present invention, physiologically acceptable salts are preferred. Physiologically acceptable salts of the compounds according to the invention can be salts of the substances according to the invention with mineral acids, carboxylic acids or sulphonic acids. Particularly preferred salts, for example, are those with hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methanesulphonic acid, ethanesulphonic acid, p-toluenesulphonic acid, benzenesulphonic acid, naphthalenedisulphonic acid, acetic acid, propionic acid, lactic acid, tartaric acid, citric acid, fumaric acid, maleic acid or benzoic acid.
Physiologically acceptable salts can likewise be metal or ammonium salts of the compounds according to the invention which have a free carboxyl group. Those particularly preferred are, for example, sodium, potassium, magnesium or calcium salts, and also ammonium salts which are derived from ammonia, or organic amines such as ethylamine, di- or triethylamine, di- or triethanolamine, dicyclohexylamine, dimethylaminoethanol, arginine, lysine or ethylenediamine.
The compounds according to the invention can be present in tautomeric forms. This is known to the person skilled in the art, and forms of this type are likewise included by the scope of the invention.
The compounds according to the invention can furthermore occur in the form of their possible hydrates.
Halogen in the context of the present invention represents fluorine, chlorine, bromine and iodine.
The compounds of the formula (I) according to the invention can be prepared by the reaction of the compound of the formula (II) 
A) with a compound of the formula (III) 
where
R1 is as defined above;
if appropriate in an organic solvent, with heating to give the compound of the formula (I);
or
B) with a compound of the formula (IV) 
where
R1 is as defined above;
in an organic solvent under heating to compounds of the formula (V) 
where
R1 is as defined above;
subsequently with a halogenating agent to compounds of the formula (VI) 
where
R1 is as defined above;
R2 represent halogen;
and finally with aqueous ammonia solution under heating and elevated pressure.
The compound of the formula (II) can be prepared according to the following reaction scheme: 
The compound of the formula (II) is obtainable in a multi-stage synthesis from the sodium salts of ethyl cyanopyruvate, which is known from the literature (Borsche and Manteuffel, Liebigs. Ann. Chem. 1934, 512, 97). By reaction thereof with 2-fluorobenzylhydrazine with heating and under a protective gas atmosphere in an inert solvent such as dioxane, ethyl 5-amino-1-(2-fluorobenzyl)pyrazole-3-carboxylate is obtained, which cyclizes by means of reaction with dimethylaminoacrolein in the acidic medium under a protective gas atmosphere and with heating to give the corresponding pyridine derivative. This pyridine derivative, ethyl 1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine-3-carboxylate, is converted by means of a multi-stage sequence, consisting of conversion of the ester into the corresponding amide using ammonia, dehydration using a dehydrating agent such as trifluoroacetic anhydride to give the corresponding nitrite derivative, reaction of the nitrile derivative with sodium ethoxide and final reaction with ammonium chloride, into the compound of the formula (II).
The compounds of the formula (III) can be prepared from the compounds t-butoxybis(dimethylamino)methane and 4-pyridylacetonitrile or 3-pyridylacetonitrile, which are commercially obtainable (e.g. from Aldrich), by reaction of these reactants, preferably in equimolar amounts, preferably at normal pressure and with stirring of the reaction solution for a number of hours, for example 2 hours, at elevated temperature, for example 60-130xc2x0 C., preferably 80-120xc2x0 C., in particular 100xc2x0 C.
The reaction of the compounds of the formulae (II) and (III) to give the compounds of the formula (I) can be carried out by use of the reactants in equimolar amounts or using the compound of the formula (III) in a slight excess in an organic solvent, for example a hydrocarbon, preferably an aromatic hydrocarbon and in particular xylene, preferably in the presence of 0.1-1 equivalent, preferably 0.3 equivalent, of a Lewis acid such as BF3Et2O or trimethylsilyl trifluorosulphonate (TMSOTf), preferably at normal pressure and with stirring of the reaction solution for a number of hours, for example 12 hours, at elevated temperature, for example 80-160xc2x0 C., preferably 100-150xc2x0 C., in particular 140xc2x0 C.
The compounds of the formula (IV) are commercially obtainable (e.g. from Mercachem) or can be synthesized by methods known to the man skilled in the art.
The conversion of the compounds of formulae (II) and (IV) to the compounds of the formula (V) can be carried out using either equimolar amounts of the reactants or a slight excess of the compound of formula (IV) in an organic solvent, for example a hydrocarbon, preferably an aromatic hydrocarbon and especially toluene, preferably under normal pressure and stirring of the reaction solution for several hours, for example 12 hours, under elevated temperature, for example 80-160xc2x0 C., especially 140xc2x0 C.
The conversion of the compounds of formula (V) to the compounds of formula (VI) can be carried outby reacting the compounds of formula (V) with a halogenating agent, optionally in an organic solvent conventionally used for such reactions, for example in dimethylformamide (DMF), preferably under normal pressure and stirring of the reaction solution for several hours, for example 3 hours, under elevated temperature, for example 80-160xc2x0 C., especially 120xc2x0 C. According to the invention the use of POCl3 as halogenating agent is preferred.
The conversion of the compounds of the formula (VI) to the compounds of the invention according to formula (I) can be carried out by reacting the compounds of the formula (VI) with aqueous ammonia solution preferably under elevated pressure, for example by carring out the reaction in an autoclave letting the reaction proceed under the pressure of the reaction solution itself, and stirring of the reaction solution for several hours, for example 12 hours, under elevated temperature, for example 80-160xc2x0 C., preferably 100-150xc2x0 C., especially 140xc2x0 C.
The compounds of the formula (I) according to the invention show an unforeseeable, valuable spectrum of pharmacological activity.
The compounds of the formula (I) lead to vasorelaxation, inhibition of platelet aggregation and to a lowering of blood pressure and to an increase in the coronary blood flow. These actions are mediated via direct stimulation of soluble guanylate cyclase and an intracellular cGMP increase. Moreover, the compound of the formula (I) according to the invention potentiates the action of substances which increase the cGMP level, for example EDRF (endothelium-derived relaxing factor), NO donors, protoporphyrin IX, arachidonic acid or phenylhydrazine derivatives.
They can therefore be employed in medicaments for the treatment of cardiovascular diseases, for example for the treatment of high blood pressure and cardiac insufficiency, stable and unstable angina pectoris, peripheral and cardiac vascular diseases, of arrthythmias, for the treatment of thromboembolic diseases and ischaemias such as myocardial infarct, stroke, transitory and ischaemic attacks, peripheral circulatory disorders, prevention of restenoses such as after thrombosis therapy, percutaneous transluminal angioplasty (PTA), percutaneous transluminal coronary angioplasty (PTCA), bypass and also for the treatment of arteriosclerosis, asthmatic conditions and diseases of the urogenital system such as prostate hypertrophy, erectile dysfunction, female sexual dysfunction, osteoporosis, gastroparesis and incontinence.
The compounds of the formula (I) described in the present invention are also active compounds for the control of diseases in the central nervous system which are characterized by disturbances of the NO/cGMP system. In particular, they are suitable for improving the perception, concentration powder, learning power or memory power after cognitive disorders, such as occur, in particular, in situations/diseases/syndromes such as mild cognitive impairment, age-associated learning and memory disorders, age-associated memory losses, vascular dementia, craniocerebral trauma, stroke, dementia which occurs after strokes (post-stroke dementia), post-traumatic craniocerebral trauma, general concentration disorders, concentration disorders in children with learning and memory problems, Alzheimer""s disease, vascular dementia, dementia with Lewy bodies, dementia with degeneration of the frontal lobes including Pick""s dementia, Parkinson""s disease, progressive nuclear palsy, dementia with corticobasal degeneration, amyolateral sclerosis (ALS), Huntington""s disease, multiple sclerosis, thalamic degeneration, Creutzfeld-Jacob dementia, HIV dementia, schizophrenia with dementia or Korsakoff psychosis. They are also suitable for the treatment of disorders of the central nervous system such as states of anxiety, tension and depression, central nervous system-related sexual dysfunctions and sleep disorders, and for the regulation of pathological disorders of the absorption of food, tea, coffee, etc. and addictive drugs.
Furthermore, the active compounds are also suitable for regulating the cerebral circulation and are thus effective agents for the control of migraine.
They are also suitable for the prophylaxis and control of the sequellae of cerebral infarcts (cerebral apoplexy), such as stroke, cerebral ischaemias and craniocerebral trauma. The compounds of the formula (I) according to the invention can likewise be employed for the control of painful conditions.
The compounds according to the invention moreover have anti-inflammatory action and can therefore be employed as anti-inflammatory agents.
The invention moreover includes the combination of the compounds of the formula (I) according to the invention with organic nitrates and NO donors.
Organic nitrates and NO donors in the context of the invention are generally substances which display their therapeutic action via the release of NO or NO species. Sodium nitroprusside, nitroglycerin, isosorbide dinitrate, isosorbide mononitrate, molsidomine and SIN-1 are preferred.
The invention moreover includes the combination with compounds which inhibit the breakdown of cyclic guanosine monophosphate (cGMP). These are, in particular, inhibitors of phosphodiesterases 1, 2 and 5; nomenclature according to Beavo and Reifsynder (1990) TiPS 11 pp. 150 to 155. By means of these inhibitors, the action of the compounds according to the invention is potentiated and the desired pharmacological effect is increased.
Biological Investigations
Vasorelaxant Action in Vitro
Rabbits are stunned by a blow to the neck and exsanguinated. The aorta is removed, freed from adherent tissue, divided into rings 1.5 mm wide and individually transferred under a pretension to 5 ml organ baths containing carbogen-aerated Krebs-Henseleit solution of the following composition (mM) at 37xc2x0 C.: NaCl: 119; KCl: 4.8; CaCl2xc3x972H2O: 1; MgSO4xc3x977H2O; 1.4; KH2PO4: 1.2; NaHCO3:25; Glucose: 10. The contractile force is determined using Statham UC2 cells, amplified and digitalized by means of A/D converters (DAS-1802 HC, Keithley Instruments Munich), and recorded in parallel on linear recorders. To produce a contraction, phenylephrine is added to the bath cumulatively in increasing concentration. After several control cycles, the substance to be investigated is investigated in each further passage in increasing dose in each case and the height of the contraction is compared with the height of the contraction reached in the last previous passage. From this, the concentration is calculated which is necessary in order to reduce the height of the control value by 50% (IC50). The standard administration volume is 5 xcexcl and the proportion of DMSO in the bath solution corresponds to 0.1%. The result is shown below in Table 1:
Determination of the Liver Clearance in Vitro
Rats are anesthetized, heparinized, and the liver is profused in situ via the portal artery. Ex vivo, the primary rat hepatocytes are then obtained from the liver by means of collagenase solution. 2xc2x7106 hepatocytes per ml were incubated at 37xc2x0 C. with the same concentration in each case of the compound to be investigated. The decrease of the substrate to be investigated over time was determined bioanalytically (HPLC/UV, HPLC/fluorescence or LC/MSMS) at 5 points in time in each case in the period from 0-15 min after the start of incubation. From this, the clearance was calculated by means of the cell count and liver weight.
Determination of the Plasma Clearance in Vivo
The substance to be investigated is administered intravenously to rats via the tail vein as a solution. At fixed points in time, blood is taken from the rats, heparinized and plasma is obtained therefrom by conventional measures. The substance is quantified bioanalytically in the plasma. The pharmacokinetic parameters are calculated from the plasma concentration-time courses determined in this way by means of conventional non-compartmental methods used for this purpose.
The present invention includes pharmaceutical preparations which, in addition to non-toxic, inert pharmaceutically suitable vehicles, contain the compounds of the formula (I) according to the invention, and processes for the production of these preparations.
The active compound can optionally also be present in microencapsulated form in one or more of the vehicles indicated above.
The therapeutic reactive compound of the formula (I) should be present in the abovementioned pharmaceutical preparations in a concentration of approximately 0.1 to 99.5% by weight, preferably of approximately 0.5 to 95% by weight, of the total mixture.
Apart from the compounds of the formula (I) according to the invention, the abovementioned pharmaceutical preparations can also contain further pharmaceutical active compounds.
In general, it has proven advantageous both in human and in veterinary medicine to administer the active compound according to the invention in total amounts of approximately 0.01 to approximately 700, preferably 0.01 to 100, mg/kg of body weight every 24 hours, if appropriate in the form of several individual doses, to achieve the desired results. An individual dose contains the active compound according to the invention preferably in amounts from approximately 0.1 to approximately 80, in particular 0.1 to 30, mg/kg of body weight.
The present invention is shown below in greater detail with the aid of non-restricting preferred examples. If not stated otherwise, all quantitative data relate to percentages by weight.